ANALYSIS OF UNSATURATED C-27 STEROLS BY NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

Complete H-1 and C-13 nuclear magnetic resonance (NMR) signal assignme nts have been established for 5 alpha-cholestan-3 beta-ol, 22 unsatura ted C-27 sterols, and their acetate derivatives. Assignments were made from a combination of 1D and 2D spectra and include stereochemical H- 1 assignments for the C-22 and C-23 protons of 5 alpha-cholesta-8,24-d ien-3 beta-ol and other Delta(24) sterols with a C-8 side chain. At th e temperature and concentration range described, chemical shifts were generally reproducible to +/-0.01 ppm in C-13 spectra and +/-0.001 ppm in H-1 spectra. Except for some overlapped or strongly coupled H-1 re sonances, chemical shifts are given to these precisions, which are an order of magnitude better than for most data given previously. Full H- 1 NMR data have been reported previously for only three of the 46 comp ounds, and C-13 data were unavailable for many, including the previous ly undescribed cholesta-5,8(14)-dien-3 beta-ol. An extensive set of H- 1 .H-1 coupling constants for the unsaturated sterols indicated consid erable conformational diversity, which was confirmed by molecular mode ling. The conformational diversity together with other factors led to a complex pattern of C-13 substituent-induced chemical shifts (SCS) th at appeared to elude any simple empirical correlations with structure. By contrast, the H-1 SCS correlated reasonably well with simple struc tural features. The high precision of the SCS revealed small but measu rable effects of a double bond on H-1 resonances up to 12 bonds away. Also discussed are the utility and limitations of NMR for the identifi cation of unsaturated sterols, estimation of purity, and analysis of m ixtures, with an emphasis on special problems encountered at a microgr am level.